Microfluidic size-exclusion devices, systems, and methods

ABSTRACT

Microfluidic devices, assemblies, and systems are provided, as are methods of manipulating micro-sized samples of fluids. Microfluidic devices having a plurality of specialized processing features are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation Application of U.S. patentapplication Ser. No. 14/137,841 filed Dec. 20, 2013, which is aContinuation of U.S. patent application Ser. No. 12/850,326 filed Aug.4, 2010, now U.S. Pat. No. 8,623,296, which issued on Jan. 7, 2014,which is a Continuation application of U.S. patent application Ser. No.11/796,783 filed Apr. 30, 2007, which is a Divisional patent applicationof U.S. patent application Ser. No. 10/336,706 filed Jan. 3, 2003, nowU.S. Pat. No. 7,214,348, which issued on May 8, 2007, which claimsbenefit under 35 U.S.C. § 119(e) from earlier filed U.S. ProvisionalPatent Application Nos. 60/398,851 and 60/398,946, both filed Jul. 26,2002, and U.S. Provisional Patent Application No. 60/399,548, filed Jul.30, 2002; all of which are herein incorporated in their entireties byreference. Cross-Reference is also hereby made to U.S. patentapplication Ser. Nos. 10/336,274 and 10/336,330, both filed Jan. 3,2003, both of which are also herein incorporated in their entireties byreference.

FIELD

The present application relates to microfluidic devices, systems thatinclude such devices, and methods that use such devices and systems.More particularly the present invention relates to devices thatmanipulate, process, or otherwise alter micro-sized amounts of fluidsand fluid samples.

BACKGROUND

Microfluidic devices are useful for manipulating fluid samples. Therecontinues to exist a demand for microfluidic devices, systems of usingthem, systems for processing them, and methods of manipulating fluids,that are fast, reliable, consumable, and can be used to process a largenumber of samples simultaneously.

SUMMARY

According to various embodiments, a microfluidic device is provided thatincludes a substrate, a first channel, a second channel, a columnconnecting the first and second channels, and a filter frit materialdisposed in the column. The substrate can have first and second opposingsurfaces and a thickness. The first channel can be formed in the firstsurface and can have a first depth extending in a direction normal tothe first surface and toward the second surface. The first depth can beequal to or less than the thickness of the substrate. The second channelcan be formed in the second surface and can have a second depthextending in a direction normal to the second surface and toward thefirst surface. The second depth can be equal to or less than thethickness of the substrate. The column can have a height that extendsfrom the first surface to the second surface. The column can have aconstant cross-sectional area and/or a constant diameter, along planesthat lie parallel to the first surface, from the first surface to thesecond surface.

According to various embodiments, an integrated gel filtration fit isprovided that includes a body comprising a form-stable filter fritmaterial, a chamber formed in the body, and a gel filtration materialdisposed in the chamber.

According to various embodiments, a microfluidic device is provided thatincludes a substrate, a first channel, a second channel, a fluidcommunication between the first channel and the second channel, and aflow-restricting particulate material piled-up or log-jammed at thefluid communication. According to such embodiments, the substrate canhave a first surface, a second surface opposing the first surface, and athickness. The first channel can be formed in the substrate and canextend in a first direction. The first channel can have a firstcross-sectional area defined by at least a first minimum dimension and afirst depth, the first depth extending in a direction normal to thefirst surface and toward the second surface. The second channel can beformed in the substrate and can extend in a second direction. The secondchannel can have a second cross-sectional area defined by at least asecond minimum dimension and a second depth, the second depth extendingin a direction normal to the first surface and toward the secondsurface. The fluid communication can be formed in the substrate betweenthe first channel and the second channel and can have a thirdcross-sectional area defined by at least a third minimum dimension,where the third cross-sectional area is less than the firstcross-sectional area. The flow-restricting material can be disposed inthe first channel, in the fluid communication, or in both the firstchannel and in the fluid communication. The flow-restricting materialcan include gel filtration particles, where at least 10% by weight ofthe flow-restricting particles comprises flow-restricting particleshaving an average particle diameter that is less than the third minimumdimension.

According to various embodiments, a microfluidic device is provided thatincludes a substrate, a first channel formed in the substrate, and afirst chamber formed in the substrate, wherein the first chamber has adepth and a teardrop-shaped cross-sectional area when cross-sectionedperpendicular to the depth. The first chamber can have a substantiallycircular first end and a narrower and opposite second end, which endscollectively define a teardrop-shaped cross-section. The cross-sectionof the first chamber can be constant along the depth of the firstchamber. The second end of the first chamber can be in fluidcommunication with the first channel.

According to various embodiments, a microfluidic device is provided thatincludes a substrate having a first surface, a second surface opposingthe first surface, and a thickness, and a plurality of parallel pathwaysformed in the substrate, wherein each of the pathways comprises an inputopening, an output opening, at least one processing chamber locatedbetween the input opening and the output opening, and wherein the inputopening, the at least one processing chamber, and the output opening ateach pathway are arranged linearly. Each of the plurality of parallelpathways can include at least one valve that is capable of beingactuated to provide a fluid communication between the at least oneprocessing chamber and at least one of the input opening and the outputopening. Each of the plurality of pathways can include at least onevalve that comprises a first deformable material having a firstelasticity, a second deformable material having a second elasticity thatdiffers from the first elasticity, and an adhesive material.

According to various embodiments, a sample processing system is providedthat includes a microfluidic device as described herein, a platen, adrive unit, and a control unit wherein the platen includes amicrofluidic device holder to hold the microfluidic device. Themicrofluidic device can have a substrate having a first surface, asecond surface opposing the first surface, and a thickness, and aplurality of parallel pathways formed in the substrate, each of thepathways comprising an input opening, an output opening, and at leastone processing chamber between and in fluid communication with the inputopening and the output opening. The platen can have an axis of rotationand the holder can be disposed spaced from, and off-center with respectto, the axis of rotation. The drive unit can be capable of rotating theplaten about the axis of rotation, and the control unit can be capableof controlling the drive unit.

According to various embodiments, a method of fabricating a microfluidicdevice is provided, wherein the microfluidic device includes asubstrate, an input opening formed in the substrate, a first channelformed in the substrate and in fluid communication with the inputopening, a second channel formed in the substrate, and a fluidcommunication between the first channel and the second channel. Themethod can include introducing a flow-restricting material through theinput opening and into the first channel, and applying centripetal forceto the microfluidic device to pack the flow-restricting material in thefirst channel at the fluid communication and to prevent a substantialportion of the flow-restricting material from moving through the fluidcommunication and into the second channel.

According to various embodiments, a microfluidic device is providedhaving a substrate, a first recess formed in the substrate, a secondrecess formed in the substrate, and an intermediate wall interposedbetween the first recess and the second recess, wherein the intermediatewall portion is formed from a deformable material having a firstelasticity. An elastically deformable cover layer is also providedcovering the first recess, and a particulate flow-restricting materialcan be disposed in the first recess. The elastically deformable coverlayer can have a second elasticity that is less than the firstelasticity, wherein the elastically deformable covered layer contactsthe intermediate wall when the intermediate wall is in a non-deformedstate, and wherein the elastically deformable cover layer does notcontact the intermediate wall when the intermediate wall is in adeformed state, thereby forming a fluid communication between the firstand second recesses. The fluid communication between the first andsecond recesses can be designed or formed as a flow restrictor asdescribed herein.

The invention may be more fully understood with reference to theaccompanying drawing figures and the descriptions thereof. Modificationsthat would be recognized by those skilled in the art are considered apart of the present disclosure and within the scope of the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top view of a microfluidic device according to an embodimentwherein a first channel is formed in a first surface, a second channelis formed in a second surface, and an interconnecting column of constantdiameter and having a frit material disposed therein;

FIG. 2 is a cross-sectional side view of the microfluidic device shownin FIG. 1 taken along line 2-2 of FIG. 1;

FIG. 3 is a top view of another embodiment of a microfluidic deviceincluding an integrated gel filtration frit;

FIG. 4 is a cross-sectional side view of the microfluidic device shownin FIG. 3 taken along line 4-4 of FIG. 3;

FIG. 5 is a top view of a microfluidic device according to an embodimentincluding an integrated gel filtration frit;

FIG. 6 is a cross-sectional side view of the microfluidic device shownin FIG. 5 taken along line 6-6 of FIG. 5;

FIG. 7 is a perspective view of an integrated gel filtration frit havinga form-stable body, a chamber in the body, and a gel filtration materialdisposed in the chamber;

FIG. 8 is a cross-sectional side view of an integrated gel filtrationflit including a form-stable body as shown in FIG. 7 being filled with agel filtration material by using a nozzle;

FIG. 9 is a perspective view of a form-stable body for use in preparingan integrated gel filtration frit and having a chamber;

FIG. 10 is a cross-sectional side view of the form-stable body shown inFIG. 9 being filled with a gel filtration material by using a nozzle;

FIG. 11 is a perspective view of a multi-nozzle machine useful infilling a plurality of integrated gel filtration frits simultaneously;

FIG. 12 is a top view in partial cross-section of a microfluidic devicethat includes a fluid communication having a conical shape, andincluding two types of particles sizes;

FIG. 13 is a top view in partial cross-section of an embodiment of aside view of a microfluidic device including a gel filtration materialthat can be used as a flow restrictor;

FIGS. 14 and 15 are top views in partial cross-section of embodiments ofa microfluidic device having baffles to restrict the flow of fluid andcause a pile-up of gel filtration particles;

FIGS. 16 and 17 are top views of various embodiment of a microfluidicdevice including a fluid communication having an abrupt change in thecross-sectional area between a first channel and a second channel;

FIG. 18 is a flowchart with corresponding cross-sectional views,depicting a method for forming a microfluidic device;

FIG. 19 is a flowchart depicting a method of preparing a microfluidicdevice for use as a purification device;

FIG. 20 is a top view of an embodiment of a microfluidic device having asubstrate, a plurality of parallel pathways formed in the substrate, anda plurality of valves for each pathway;

FIG. 21 is a perspective view of an embodiment of a substrate having aplurality of pathways;

FIG. 22 is a top view of an embodiment that includes a plurality ofteardrop-shaped chambers arranged on a cant and formed in a substrate;

FIG. 23 is an enlarged perspective view of an embodiment of ateardrop-shaped input chamber having a tapering cross-section;

FIG. 24 is a top view of a microfluidic device according to anembodiment having a pathway for processing a sample;

FIG. 25 is an enlarged perspective view of the pathway shown in thedevice of FIG. 24;

FIG. 26 is a perspective view of an embodiment of a microfluidic systemcomprising microfluidic devices held on a rotatable platen that can berotated by a drive unit, heated by a heating element, and controlled bya control unit;

FIGS. 27a-27d are cross-sectional views of a microfluidic channel havingvarious profiles in the substrate;

FIG. 28 is an exploded perspective view of an assembly including asample processing device and a carrier;

FIG. 29 is a perspective view of the assembly of FIG. 28 as assembled;

FIG. 30 is an enlarged view of a portion of a carrier depicting one setof main conduit support rails and collars useful in isolating theprocess chambers on a sample processing device;

FIG. 31 is a partial cross-sectional view of a portion of a carrierillustrating an example of a force transfer structure useful within thecarrier;

FIG. 32 is a partial cross-sectional view of a carrier and sampleprocessing device assembly including an optical element in the carrier;

FIG. 33 depicts a carrier and sample processing device assemblyincluding an alignment structure for a sample processing deliverydevice;

FIG. 34 is an exploded perspective view of another sample processingdevice and carrier assembly according to various embodiments; and

FIG. 35 is a block diagram of a thermal processing system that can beused in connection with sample processing devices.

Other various embodiments of the present invention will be apparent tothose skilled in the art from consideration of the specification andpractice of the devices, systems, and methods described herein, and thedetailed description that follows. It is intended that the specificationand examples be considered as exemplary only, and that the true scopeand spirit of the invention includes those other various embodiments.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

FIG. 1 is a top view of a microfluidic device 98 according to variousembodiments that include a substrate 100, an input opening 102, anoutput opening 110, a first channel 104, a second channel 108, a chamber106 interconnecting the first channel 104 and the second channel 108,and a filter frit material 112 disposed in the chamber 106. The chamber106 can be in the form of a column, for example, a vertical cylindricalcolumn as shown.

FIG. 2 is a cross-sectional side view of the microfluidic device 98 ofFIG. 1, taken along line 2-2 of FIG. 1. As shown in FIGS. 1 and 2,covers 114, 116, and 118 are provided in contact with the substrate 100.Cover 114 covers the bottom, as shown in FIG. 2, of the substrate 100and provides an inner surface 115 that can, in-part, define channel 104.A fluid sample introduced in input opening 102 can pass from inputopening 102 into first channel 104, through first channel 104 and intochamber 106, through filter flit material 112 in chamber 106 and intosecond channel 108, and from second channel 108 into output opening 110.First channel 104 can be loaded with a gel filtration material (notshown), for example, an ion-exchange gel filtration material.

Input opening 102 can be designed as an entry port, a hole through alayer, an aperture, or any other feature that provides an entrance to achannel or chamber in fluid communication therewith. Output opening 110can be designed as a port, aperture, a hole through a layer, or anyother feature that provides an exit from a channel or chamber in fluidcommunication therewith. Input opening 102 and/or output opening 110 canbe covered or partially covered by a frangible or puncturable materialcover 116,118 that can be in the form of a tape, a film, a sheet, amembrane, or a combination thereof. Cover 114 for the bottom (as shown)of the device can be a tape, a film, a sheet, a membrane, or acombination thereof. Any of covers 114, 116, and 118 can be in the formof a second substrate affixed to, secured to, bonded to, or otherwiseconnected to the substrate 100. First channel 104, second channel 108,chamber 106, or combinations thereof can be pro-filled with reagents,reactants, or buffers known in the art, before the respective cover isapplied to substrate 100. Additionally, first channel 104, secondchannel 108, chamber 106, or combinations thereof can be loaded throughthe input opening.

FIG. 3 is a top view of a microfluidic device 498 that includes a filterfrit material 412 having a shape that complements the shape of a column406 in which the filter frit material 412 is disposed. The filter fritmaterial 412 can include a chamber 413 that retains a gel filtrationmaterial 418. FIG. 4 is a side view of the microfluidic device 498 shownin FIG. 3. In the embodiment shown in FIGS. 3 and 4, the device furtherincludes a substrate 400, an input opening 402, a first channel 404, achamber 406 for accommodating filter frit material 412, a second channel408, and an output opening 410. The device 498 shown in FIGS. 3 and 4can also include a first cover 414, and a second cover 416. The filterflit material 412 can have an outer shape that is complementary to theinner shape of chamber 406.

FIGS. 5 and 6 show an embodiment of a microfluidic device 700 thatincludes a substrate 701 and a filter frit material 712 having a shapethat complements a chamber 713. An opening 720 of the filter flitmaterial 712 faces an input opening 702 formed in substrate 701. Thefilter frit material 712 further includes a closed end 722 orientedtowards an output opening 710 formed in a substrate 701. The filter fritmaterial 712 can be filled with a filtration material 718, for example,an ion-exchange gel filtration material. Covers 714 and 716 can besecured, bonded, adhered, or otherwise affixed to substrate 701 using anadhesive 715. The adhesive can be, for example, a pressure sensitiveadhesive.

The microfluidic devices 98, 498, and 700 shown in FIGS. 1-6 can be usedfor filtering liquids that are manipulated to pass through the devices.The devices can be used, for example, for gel filtration, size-exclusionfiltration, ion-exchange filtration, or combinations of these filtrationtechniques. For example, filtration materials can be loaded and/orincluded in the devices, and can include small beads of filtrationmaterials. Size-exclusion materials can be used that can retain smallermolecules of an aqueous sample while allowing larger molecules of thesample to pass through or around. For example, P-10 BIO-GEL materialsfrom Bio-Rad can be used and are composed of acrylamide particles thatare roughly 45-90 μm in average particle size diameter. These particles,when hydrated, can capture free dyes, undesired nucleotides, and saltions from a sample as the sample migrates through the materials.

Samples can be manipulated through devices 98, 498, and 700 by gravitypressure differentials, or centripetal force, for example. The resultingfiltrates that elute from the devices can then be analyzed, used, orsubsequently passed on through the device to a subsequent stage ofprocessing, for example, into a PCR reaction chamber, a sequencingreaction chamber, or other processing reaction chamber.

According to various embodiments, the filter frit material 112, 412, or712 shown in FIGS. 1-6 can be “press fit” into the respective chamber,placed in the respective chamber, or otherwise positioned in therespective chamber.

The covers described above with reference to FIGS. 1-6 can include aplastic material, for example, a polyolefin material. According tovarious embodiments, covers can include tape or film materials coatedwith a pressure-sensitive adhesive, or plastic materials that can bethermally bonded to a respective substrate.

According to various embodiments including those shown in FIGS. 1-6, thedevice can include one or more channels that can be rectangular incross-sectional shape. The devices can include channels that can be, forexample, from about 0.1 mm to about 1.0 cm deep, from about 0.1 mm toabout 1.0 cm wide, and from about 0.1 mm to about 10.0 cm long. Anexemplary channel can be 0.50 mm deep, 0.50 mm wide, and 20 mm long,thus providing total volume of about 5 μL.

According to various embodiments including those of FIGS. 1-6, a gelfiltration material can be disposed in a channel of the device. The gelfiltration material can be loaded into the device by pipetting into aninput opening of the device and/or drawing the material into the deviceby using vacuum force, for example, applied to an output opening of thedevice. A channel of the device can be filled with a gel filtrationmaterial by pressure loading the gel filtration material through aninput opening of the device to dispense the gel filtration material in achannel or chamber of the device. In an exemplary embodiment, a fullyhydrated gel filtration material is loaded into a channel of the device,for example, into first channel 104 of device 98 of FIG. 1. Once thechannel is filled with hydrated gel filtration material, the device canbe centrifuged to de-water the gel filtration material and to “pack” thegel filtration material, forming a purification column. This process canbe used to prepare the device for sample filtration and can be used toremove unnecessary or excess water or buffer from the gel filtrationmaterial. In a variant of this process, the excess water or buffer canbe collected in an outlet channel or chamber and later used to diluteand increase the volume of a filtered sample to render the sampleinjection-ready.

According to various embodiments including the embodiments of FIGS. 1-6,the device can include additional chambers and/or channels. For example,the device can include a PCR amplification chamber, a sequencingreaction chamber, or both a PCR amplification chamber and a sequencingreaction chamber. According to various embodiments, the device caninclude an output chamber useful for holding a sample prior to injectingthe sample into a sequence detection system or other analyticaldetector.

According to various embodiments, microfluidic devices are provided thatinclude a plurality of sample processing pathways as described herein,in a single device.

According to various embodiments, a porous filter frit material can beused to prevent the gel filtration material loaded in a channel fromflowing out of the channel. The average pore size of the filter fritmaterial can be chosen to allow fluids to pass through (water, sample,etc.) while constraining the movement of a gel filtration material suchas acrylamide beads. For instance, the microfluidic device shown in theFIGS. 1-6 can utilize a hydrophilic polyethylene filter frit materialwith an average pore size of about 33 microns. When used with P-10BIO-GEL gel filtration materials, such a frit can adequately constrainthe gel filtration materials while allowing water and sample fluids topass therethrough. An exemplary porous filter frit material that can beused for such purpose is a sintered, high-density polyethylene (HDPE)frit having a suitable average pore size.

According to various embodiments, a gel filtration retention mechanismcan be provided in a device and includes a flow-restrictor in the formof a small channel or serpentine path formed in the substrate and whichprevents the gel filtration material from passing.

According to various embodiments including the embodiments of FIGS. 1-4,wherein two channels are provided and separated by a filtration chamberor column, one of the channels can be formed in a first surface of asubstrate and a second channel can be formed in the opposing surface ofthe substrate. For example, the second channel 108 (FIG. 2) or 408 (FIG.4) can be formed in a first surface of the substrate and can providefluid communication between a processing chamber and a respective outputopening.

The second channel can have dimensions similar to, or the same as, thedimensions of the first channel, as illustrated in FIGS. 3 and 4. Thesecond channel can have a depth of from about 0.1 mm to about 1.0 cm, awidth of from about 0.1 mm to about 1.0 cm, and a length of from about0.1 mm to about 10.0 cm. An exemplary second channel has a depth ofabout 0.50 mm, a width of about 0.75 mm and a length of about 3.0 mm.The second channel can be at least partially defined by a cover, forexample, cover 116 shown in FIGS. 1 and 2, or FIG. 416 shown in FIGS. 3and 4. Regardless of whether the device includes a first channel filledwith a gel filtration material, the second channel of the device can beprovided with a gel filtration material loaded therein. Gel filtrationmaterial can be loaded into the second channel before, after, or at thesame time that a filtration frit is positioned within the device.

According to various embodiments, the output opening 110, 410, 710 canserve to capture or retain a processed sample after the sample passesthrough a processing chamber in the microfluidic device. Initially, theoutput opening 110, 410, 710 can be open so that vacuum can be appliedto the device for loading a gel filtration material. The output openingcan remain open during centrifugation of the microfluidic device tofurther pack and/or dewater the gel filtration material. During such apacking process, excess water or buffer can be purged from the deviceand can escape the device through the outlet opening 110, 410, 710. Whena sample is manipulated through the microfluidic device, as bycentrifugation, for example, the outlet opening 110, 410, 710 can besealed with a cover film 116, 416, 716 to prevent the sample from beinglost, or to otherwise retain the sample in the device.

According to various embodiments, a microfluidic device can be providedwith a plurality of pathways formed in a substrate, with each pathwaybeing similar to one of the pathways shown in FIG. 1-6. Using channelsand chambers having widths of about 0.50 mm or less, for example, it ispossible to provide up to 96 or more such pathways in the substrate andto provide a resulting substrate size equivalent to that of a standardmicro-titer tray, for example, a length of about 4.75 inches and a widthof about 3.25 inches. An exemplary device of such design is shown inFIG. 20 and incorporates the 5 μL gel filtration columns.

According to various embodiments, a microfluidic device is providedsimilar to those shown in FIGS. 1-4 and having a substrate that includesa thickness that is greater than the sum of the depth of the firstchannel and the depth of the second channel. According to variousembodiments, a microfluidic device is provided similar to the deviceshown in FIGS. 5 and 6 and having a substrate that includes a thicknessthat is the same as the depth of the filter chamber 713.

According to various embodiments, including the embodiments of FIGS.1-6, a microfluidic device is provided wherein the filtration fritmaterial 112, 412, 712 has an outer peripheral shape, the chamber 106,406, 713 has an inner peripheral shape, and the outer peripheral shapeis complementary to the inner peripheral shape.

FIGS. 7 and 9 depict exemplary embodiments of an integrated gelfiltration frit 750, 950 that includes a body 712, 772 made up of aform-stable frit material and that defines a chamber 721, 921 and anopening 728, 928. The chamber 721, 921 is filled with a gel filtrationmaterial 778, 978 that has been loaded in the chamber 721, 921. The gelfiltration flit 750, 950 can be made by a method as depicted in FIGS. 8and 10, respectively.

FIGS. 8 and 10 illustrate a nozzle 130 in the process of filling theintegrated gel filtration frits 750, 950 with a diluent 132 and a gelfiltration material 778, 978 via the opening 728, 928.

FIG. 11 depicts an embodiment of a multi-nozzle filling machine 140 forsimultaneously filling a plurality of integrated gel filtration frits.

According to various embodiments, an integrated gel filtration frit 750,950 can be formed as illustrated in FIGS. 8 and 10 and subsequentlypacked into a microfluidic device, for example, a device as shown inFIGS. 3-6. For instance, a slurry of hydrated P-10 BIO-GEL particles canbe pumped into a porous form-stable frit body, and the resulting fritcan be assembled into a microfluidic device. Such a manufacturingprocedure can reduce the number of substrate manipulations involved withforming the microfluidic device, allow for off-line filtration flitfabrication, and can reduce the overall manufacturing costs for themicrofluidic device.

According to various embodiments, the body and/or chamber of theintegrated gel filtration frit can be constructed in a rectanguloid or acylindrical shape, and the chamber can be pre-filled with more than onetype of gel using a single nozzle or multiple nozzles.

The integrated gel filtration frit can retain a gel filtration materialtherein yet allow water and liquid samples to flow therethrough.

According to various embodiments, an integrated gel filtration frit canbe provided that includes an opening in a frit body which is in fluidcommunication with an interior gel filtration material chamber.According to various embodiments, an integrated gel filtration flit canbe provided having a gel filtration material that includes anion-exchange gel filtration material. According to various embodiments,an integrated gel filtration flit can be provided having a form-stablefilter frit body that includes a porous hydrophilic polyethylenematerial. The body could also be formed using a membrane or other filtermaterials, and does not necessarily have to be form-stable. Theintegrated gel filtration flit can have a length dimension, a widthdimension, and a depth dimension, wherein each of the dimensions is lessthan 50 mm.

According to various embodiments, a microfluidic device is providedhaving a channel formed in a substrate and an integrated gel filtrationflit disposed in the channel, for example, as shown in FIG. 6. Accordingto various embodiments, a microfluidic device can be provided having achannel formed in a substrate, an input opening formed in the substrate,an output opening formed in the substrate, a filtration column orchamber formed in the substrate between and in fluid communication withthe input opening and the output opening, and an integrated gelfiltration frit as described herein disposed in the column, wherein theinput opening of the channel is in fluid communication with the openingof the integrated gel filtration frit.

FIGS. 12-16 depict various microfluidic devices 200, each of which isdesigned with one or more features for restricting the flow of afiltration material through the device. In FIGS. 12 and 14-16, thefeatures are formed in a substrate 220. In the device of FIG. 13, thechannels can be formed in an insertable component that can beincorporated into a microfluidic device. In each device, a first channel208 is in fluid communication with a second channel 210. A fluidcommunication 212 in the form of a region is provided in each devicebetween the first channel 208 and the second channel 210. In FIGS. 12and 14-16, the fluid communications 212 are also formed in the substrate220. Each of FIGS. 12-16 shows flow-restricting material 202 disposed inthe first channel 208 and/or the fluid communication 212. As shown inFIGS. 12 and 13, a second material 204 of smaller average diametricalparticle cross-section area than material 202 is provided in firstchannel 208. In FIG. 13, a third material 206 having an even smalleraverage diametrical particulate cross-sectional area is provided infirst channel 208. Each of the materials 202, 204, and 206 can include agel filtration material, such as, an ion-exchange gel filtrationmaterial. Each of the materials 202, 204, and 206 can be an inertmaterial having an average diametrical particle cross-sectional area asdescribed herein, for example, glass or silicon seeds. The microfluidicdevice 200 can also have baffles 214 as depicted in FIGS. 14 and 15, tofurther restrict the flow of the flow-restricting material 202 into thesecond channel 210. The baffles 214 can be provided in the fluidcommunication 212, in the second channel 210, or in both the fluidcommunication 212 and in the second channel 210.

Some of the particles of materials 202, 204, and 206 can flow into thesecond channel 210 before a pile-up of the materials 202, 204, and 206forms at fluid communication 212. The forming of the pile-up and/or thebreakdown of the pile-up at the fluid communication 212 can bemanipulated by controlling how much force is applied to the microfluidicdevice, for example, a centripetal force, or a pneumatic force. Thefluid communication 212 can be a tapered transition region, for example,a funnel-shaped transition region. The fluid communication 212 can be aconically-shaped transition region as depicted in FIGS. 12-15.

According to various embodiments, a method to form a microfluidic device200 as depicted in any one of FIGS. 12-15 is provided. Particulateflow-restricting material 202 is disposed in a first channel 208 havinga first cross-sectional area. The first channel 208 terminates at afluid communication 212 in the form of a region. The particles of thefirst material 202 can have an average diametrical particlecross-sectional area that is from 5% to about 90% of the diametricalcross-sectional area of the second channel 210. According to variousembodiments, an additional material 204 made-up of particles having asmaller cross-sectional area than the first material particles can thenbe added to the pile-up of the first material particles 202. A thirdtype of material 206 can be added after particulate material 204, thethird material 206 can be another flow-restricting particulate material,can be of the same composition but of smaller size than eitherparticulate material 202 or 204, or can be a gel or resin material thatcan be non-particulate. Diluent initially accompanying or used to loadthe first and/or second material can be removed from the first channel208 through the fluid communication 212 and into the second channel 210,for example, by using centripetal force. The diluent can further beremoved from the second channel 210 and, for example, removed from thedevice or stored in a collection or output chamber.

According to various embodiments, the particulate materials 202, 204,and 206 can be gel filtration particles or other particles. Theparticles can be chemically derivitized or physically modified toprovide functions other than restricting flow of subsequently loaded gelor resin materials. For example, the materials 202, 204, and 206 can bemodified to allow hybridization with DNA or DNA fragments. In caseswhere any of materials 202, 204, or 206 are modified to allowhybridization, methods can be provided whereby hybridized components cansubsequently be released from the materials, for example, by denaturing.As such, various embodiments can provide a purification or concentrationof hybridizable components.

Because the microfluidic devices 200 shown in FIGS. 12-16 can beassembled in place, methods of making the devices can avoid access andhandling problems associated with using filtration frits known in theart. For example, the devices 200 can be made smaller than devicesincorporating frits known in the art.

According to various embodiments, microfluidic devices such as thoseshown in FIGS. 12-16 can be provided wherein the direction of the flowof a fluid through the first channel is aligned with the direction offlow of fluid through the second channel. According to variousembodiments, microfluidic devices can be provided wherein at least oneof the first channel and second channel can include a cross-sectionalarea orthogonal to the direction of fluid flow, that has a round shape,for example, a circular cross-section.

According to various embodiments, for example, the embodiments shown inFIGS. 12-16, a microfluidic device can be provided that includes asubstrate having a first surface, a second surface opposing the firstsurface, and a thickness. The substrate includes a first channel formedtherein that extends in a first direction and that has a firstcross-sectional area defined by at least a first minimum dimension andfirst depth, the first depth extending in the direction normal to thefirst surface and toward the second surface. The substrate also includesa second channel formed therein and extending in a second direction,wherein the second channel has a second cross-sectional area defined byat least a second minimum dimension and a second depth. The second depthextends in a direction normal to the first surface and toward the secondsurface. The device further includes a fluid communication formed in thesubstrate between the first channel and the second channel, and having athird cross-sectional area defined by at least a third minimumdimension, wherein the third cross-sectional area is less than the firstcross-sectional area. The device further includes a particulateflow-restricting material disposed in the first channel and comprisingflow-restricting particles, wherein at least 10% by weight of theflow-restricting particles includes flow-restricting particles having aparticle diameter that is less than the third minimum dimension.According to various embodiments, the first direction and the seconddirection can be aligned with one another at the fluid communication.According to various embodiments, at least one of the first channel andthe second channel includes a cross-section that has a round shape.According to various embodiments, at least 50% by weight of theflow-restricting particles includes flow-restricting particles having aparticle diameter that is less than the third minimum dimension. Forexample, at least 95% by weight of the flow-restricting particlesincludes flow-restricting particles having a particle diameter that isless than the third minimum dimension. According to various embodiments,the flow-restricting particles have particle diameters that are lessthan the second minimum dimension. According to various embodiments, theflow-restricting material can include a gel filtration material disposedin the first channel and having an average diametrical cross-sectionalarea that is less than the third cross-sectional area. The averagediametrical cross-sectional area of the flow-restricting particles canbe from about 0.1 to about 0.2 times the third cross-sectional area.According to various embodiments, the flow-restricting particles canform a pile-up at the fluid communication. The flow-restricting materialcan include a first flow-restricting material having particles of afirst average diameter packed-together at the fluid communication, and asecond flow-restricting material having particles of a second averagediameter packed-together in the first channel and adjacent thepacked-together first flow-restricting material, and wherein the averagediameter of the first flow-restricting material particles is greaterthan the average diameter of the second flow-restricting materialparticles. Further, the second packed-together flow-restricting materialcan be spaced further from the second channel than the packed-togetherfirst flow-restricting material.

As shown in FIGS. 16 and 17, microfluidic devices can be provided thatinclude a fluid communication 212 between a first channel 208 and asecond channel 210, in the form of an abrupt change in cross-sectionalareas.

As exemplified in FIGS. 16 and 17, according to various embodiments, itmay be desirable to throttle the flow of fluid or particulate materialthrough the device, to meter the distribution of reagents, and/or toblock the flow of particulate material in a microfluidic device. Undersuch circumstances, it can be useful to employ a flow restrictoraccording to various embodiments. According to various embodiments, achannel with a cross-sectional area that is significantly smaller than aconnecting channel can be used to form a flow restrictor. Depending uponthe desired results and restriction, the dimensions of the restrictioncan be selected, for example, to retain smaller particles in the largercross-sectional area connecting channel. FIG. 17 shows representativegeometries of flow restriction designs that can be used.

According to various embodiments, one or more flow restrictor can beused to prevent the flow of gel filtration particles and/orsize-exclusion media into a connecting channel, processing chamber, oroutput well. The smaller channel can be large enough, however, to allowsample fluids to readily pass through. For example, according to variousembodiments, a first channel can include an output end having a firstcross-sectional area, and which intersects a second channel having asecond cross-sectional area that is from about 5% to about 50% thecross-sectional area of first channel. The cross-sectional area of thesecond channel can be, for example, from about 6% to about 30% of thecross-sectional area of the first channel, for example, from about 10%to about 15% of the cross-sectional area of the first channel. In anexemplary embodiment, a first channel has a square cross-section with awidth of about 0.50 mm and a depth of about 0.50 mm. A second channel influid communication with the first channel can be provided with a squarecross-section having a width of about 0.18 mm and a depth of 0.18 mm. Insuch a flow restrictor design, the cross-sectional area of the secondchannel is about 13% of the cross-sectional area of the first channel.Such a flow restrictor design can be useful in restricting the passageof gel filtration particles that have a minimum dimension of about 0.001mm or greater, for example, about 0.01 mm or greater, and can be usefulin causing a pile-up of gel filtration particles at the transitionbetween the two channels, wherein the gel filtration particles haveaverage cross-sectional areas that are smaller than the cross-sectionalarea of the second channel, as depicted in FIG. 16.

In such devices, a shoulder is provided at the intersection of the firstchannel 208 and second channel 210, and the shoulder can beperpendicular to the direction of flow of fluid through the first andsecond channels.

According to various embodiments, a flow restrictor second channel, suchas second channel 210 in FIGS. 12-17, can be formed by opening a valve,to form a fluid communication between two or more first channels orchambers, and a second channel. The dimensions of the intersection ortransition between the second channel and one or more first channelsdefines a flow restrictor as described herein. The fluid communicationcan be useful for causing a pile-up of gel-filtration at the fluidcommunication formed by the opening of a valve. Such valves and saidvalving techniques can include those described in concurrently filedU.S. patent application Ser. No. 10/336274 to Bryning et al., entitled“Microfluidic Devices, Methods, and Systems”, which is hereinincorporated in its entirety be reference.

FIG. 18 illustrates a manufacturing process for forming a microfluidicdevice, for example, the device of FIGS. 1 and 2. In a first step, asubstrate is formed that includes an input, an output, first and secondchannels, and a filtration frit column. In a second step, a filtrationfrit is positioned within the filtration frit column. Positioning can beaccomplished by press-fitting the filtration flit into the column, ordepending upon tolerances, the filtration flit can simply be droppedinto the column. In a third step, the bottom surface of the device issealed with a cover, for example, by applying a pressure-sensitiveadhesive tape to the bottom surface of the substrate. In a fourth stepof the method, the top of the filtration frit column is sealed and halfof the input and output openings are sealed.

FIG. 19 depicts a method of fabricating a microfluidic device, such asthe device of FIGS. 1 and 2. In a first step, a gel slurry includingflow-restricting particles can be filled in a first channel of a devicethrough an input opening. A force can be applied to the device to packthe gel slurry by using, for example, a vacuum at an output opening ofthe device, or by applying centripetal force to the device. The forcecan move the gel slurry from the input opening into the first channel.The input opening can be completely sealed after loading the gel slurryby applying a cover, or sealing can be affected after the first channelis packed. After the first channel has been packed, the gel slurry canbe dewatered and a cover can be applied to seal the output opening.Thereafter, the device can receive a sample for processing. Force canthen be applied to the microfluidic device to manipulate the sample tomove from the input opening to the output opening.

According to various embodiments, flow-restricting material, gelfiltration material, and sample can all be introduced through the inputopening of the device. At any of various times during the process, theinput opening can be completely sealed on a first surface of the devicewith a cover, for example, an optically transparent adhesive cover. Theinput opening can also be completely or partially sealed on an opposingsecond surface of the device. This allows for the containment of smallsamples, for example, samples sizes of from about one nanoliter to about10 μL, for example, from about 100 nanoliters to about 0.5 μl, that canbe pipetted into the input opening.

The various devices and methods described above can be implemented indevices and methods for high-throughput processing of a plurality ofsamples simultaneously. An example of such a high-throughput device isshown in FIG. 20. FIG. 20 is a top view of a microfluidic device 400having a plurality of pathways 300, each for processing a respectivesample according to various embodiments. The plurality of pathways 300can be parallel to each other. Each pathway 300 can have an inputopening 372 in interruptable and/or openable fluid communication with aplurality of respective processing chambers 376, 378, 381, 383, and 385.Each pathway 300 can terminate at a respective output opening 387, 389,as shown.

In the device of FIG. 20, each pathway 300 can include, in addition tothe processing chambers 376, 378, 381, 383, and 385, valves 391, 393,and 397. In various embodiments, each pathway can also include a flowsplitter 395 that can divide each pathway 300 into two respectivesub-pathways, such as a reverse sequencing reaction pathway and aforward sequencing reaction pathway, with each sub-pathway leading to aseparate output chamber or reservoir 387, 389, respectively.

FIG. 21 is a top view of another embodiment of a microfluidic deviceaccording to various embodiments and having a plurality of pathways 422.According to the exemplary embodiment shown in FIG. 21, each pathway 472can respectively include an input well 424, a PCR chamber 426, a PCRchamber valve 428, a PCR purification chamber 430, a PCR purificationchamber valve 432, a PCR purification chamber appendix 434, a furtherreaction input well 436, a sequencing chamber 438, a sequencing chambervalve 440, a sequencing purification chamber 442, and an output well,chamber, or reservoir 444, all formed in a substrate 420. Exemplaryvalves that can be useful in this and other various embodiments includethe valves described in U.S. Provisional Patent Application No.60/398,851 to Bryning et al., which is incorporated herein in itsentirety by reference.

FIGS. 22 and 23 are enlarged views of the input chamber of a device,such as the devices shown in FIG. 21, and show a plurality ofteardrop-shaped chambers 250 formed in a substrate 260. Theteardrop-shaped chamber 250 can each have a substantially circular firstend 252, a narrower and opposite second end 256, and an opening 258 influid communication with a channel 254 that leads to a subsequentfeature, such as a processing chamber, of the device.

According to various embodiments, teardrop-shaped chamber 250 can have aconstant cross-sectional area along the depth of the chamber. Accordingto various embodiments, the bottom of the teardrop-shaped chamber can bescalloped, or it can be flat.

Because the centripetal force exerted on rectilinear devices is notnecessarily aligned with each pathway, channel, well, or chamber of suchdevices, a dead volume zone can be created in comers of such. Accordingto various embodiments, to facilitate the complete transfer of samplesand prevent portions of sample from being retained, the teardrop-shapedchamber 250 can be employed to direct the sample into the connectingchannel 254. This design can be used for all non-radial wells, both tothe left or to the right of the center of the device. FIG. 22 depicts anexemplary pattern of such wells.

According to various embodiments, the teardrop-shaped chambers can becanted or rotated 45° with respect to the direction of sample flowthrough the pathways, to improve the transfer of the samples into andthrough the pathways. The direction of the cant can depend on thelocation of the wells with respect to the axis of rotation of the deviceor of a spinning platen on which the device is held, mounted, affixed,or secured.

According to various embodiments, methods are provided to manipulate aliquid sample in a microfluidic device having a teardrop-shaped chamberand a liquid sample disposed in the chamber. The device can be spunaround an axis of rotation that does not lie on any portion of thedevice. The spinning can centripetally force the liquid sample from thechamber into a channel. Methods are also provided for centripetallymanipulating a liquid sample in a channel into a chamber.

FIG. 24 is a top view of a microfluidic device having a pathway 300 forprocessing a sample according to various embodiments. FIG. 25 is anenlarged top view of the pathway 300 shown in FIG. 24. The pathway 300is exemplary of the pathways 300 shown in FIG. 20. The pathway 300 caninclude an input chamber 302, an input channel 304, a PCR chamber 306, aPCR chamber valve 308, a PCR purification column 310, a PCR purificationcolumn valve 312, a flow splitter 334, a flow splitter valve 314, aforward sequencing reaction chamber 315, a reverse sequencing reactionchamber 316, sequencing reaction chamber valves 318, 319, a forwardsequencing reaction purification column 323, a reverse sequencingreaction purification column 320, a forward sequencing reaction columnvalve 321, a reverse sequencing reaction column valve 322, a forwardsequencing reaction product output chamber 326, and a reverse sequencingreaction product output chamber 324. The device depicted in FIG. 24 isshown also including a substrate 368 and a cover 360.

According to various embodiments, the channels, chambers, valves andother components of a microfluidic device with parallel pathways can bespaced, for example, 9 mm, 4.5 mm, 3 mm, 2.25 mm, 1.125 mm, or 0.5625mm, from one another. The pathways 300 can be parallel, and can bearranged and mounted on a rotating platen so as not to lie on a radiusof rotational motion. Further details regarding microfluidic deviceshaving geometrically parallel processing pathways, and systems andapparatus including such devices or for processing such devices, aredescribed in concurrently filed U.S. patent application Ser. No.10/336274 to Bryning et al. entitled “Microfluidic Devices, Methods, andSystems”, and in concurrently filed U.S. patent application Ser. No.10/336330 to Desmond et al. entitled “Micro-Channel Design Features ThatFacilitate Centripetal Fluid Transfer”, both of which are hereinincorporated in their entireties by reference.

According to various embodiments, the device can be loaded with apipette. Prior to injecting a sample, the device can be pre-loaded withappropriate reactants, reagents, buffers, or other conventionally knowncomponents useful for carrying out desired reactions in the device.

According to various embodiments, the microfluidic device can be alaminated, multi-layer polymeric material device that can conform to anSBS microplate format. The microfluidic device can be about 0.5 mm toabout 5 mm thick, for example, from about 2.0 mm to about 3.0 mm thick.In its basic form, the microfluidic device can include a substrate thatis laminated on both sides with thin cover films. Within the substratecan be a series of channels, chambers, and/or wells that can be used tomanipulate a sample fluid along a prescribed path. Fluid samples can betransferred from channel or chamber to channel or chamber by centripetalforce. Centripetal force can be generated by rotating the device aboutan axis of rotation while mounted on a spinning platen. Thus, samplefluid can be transferred from one end of the device to the other asvarious reactions are sequentially performed.

The device can be rotated such that the fluid moves through the deviceunder centripetal force even if the entire pathway of the device issealed.

According to various embodiments, a processed sample resulting from theuse of the microfluidic device, can be an aqueous, injection-readysample that can be used in a capillary electrophoresis analyticalinstrument, for example.

According to various embodiments, the device can include a purificationcolumn that can enable the purification of small volumes, for example,volumes of from about one nanoliter to about 10 μl, for example, fromabout 100 nanoliter to about 0.5 μl. Various embodiments enable thepurification of small sample volumes in a high-throughput, parallel,planar format.

According to various embodiments, a microfluidic device is providedhaving a rectangular substrate.

According to various embodiments, a microfluidic device is provided witha pathway having a first channel and a first chamber at least partiallyformed in a substrate, and wherein the substrate includes a plurality ofsuch pathways. Each respective chamber has a depth and a teardrop-shapedcross-sectional area when cross-sectioned perpendicular to the depth.The respective chambers each have substantially circular first end, anda narrower and opposite second end. The second ends of the respectivechambers are in fluid communication with the respective channels.According to various embodiments, a microfluidic device as such isprovided having a plurality of such pathways arranged parallel to oneanother.

According to various embodiments, a microfluidic device is provided thatincludes a plurality of parallel sample processing pathways, and atleast one valve along each pathway. The at least one valve can include,a first recess formed in a substrate, a second recess formed in thesubstrate, and an intermediate wall interposed between the first recessand the second recess, wherein the intermediate wall portion is formedfrom a deformable material having a first elasticity. The valve can alsoinclude an elastically deformable cover layer covering the first andsecond recesses and having a second elasticity that is greater than thefirst elasticity, in other words, the cover layer can be more elastic orcan rebound faster, than the intermediate wall material. The elasticallydeformable cover layer can contact the intermediate wall when theintermediate wall is in a non-deformed state, and can be out of contactwith the intermediate wall when the intermediate wall is in a deformedstate, thereby forming a fluid communication between the first andsecond recesses.

Further details of such valves can be found in U.S. ProvisionalApplication No. 60/398,851 filed Jul. 26, 2002, and in concurrentlyfiled U.S. patent application Ser. No. 10/336,274, to Bryning et al,entitled “Microfluidic Devices, Methods, and Systems”, which are bothincorporated herein in their entirety by reference.

According to various embodiments, a microfluidic device is provided thatincludes a plurality of parallel processing pathways and at least onevalve along each pathway, where the at least one valve includes a firstrecess formed in a substrate and including a first recess portion and asecond recess portion. The first recess is at least partially defined byopposing wall surface portions. The opposing wall surface portionsinclude a first deformable material having a first elasticity. The firstrecess portion and the second recess portion are in fluid communicationwith each other when the first deformable material is in a non-deformedstate. The at least one valve also includes an elastically deformablecover layer having a second elasticity that is greater than the firstelasticity, in other words, the cover layer can be more elastic or canrebound faster, than the deformable opposing wall surface portion. Thecover layer covers at least the first recess portion. The opposing wallsurface portion that comprises the first deformable material isdeformable to form a barrier wall interposed between the first recessportion and the second recess portion, and to prevent fluidcommunication between the first recess portion and the second recessportion when the barrier wall is in a deformed state.

According to various embodiments, the substrate of the microfluidicdevice can include a single layer of material, a coated layer ofmaterial, a multi-layered material, or a combination thereof. Anexemplary substrate can include a single-layer substrate of a hardplastic material, such as a polycarbonate material. Materials that canbe used for the microfluidic device or a component thereof, for example,a substrate, base layer, recess-containing layer, or any combinationcomponents, can include polycarbonate, polycarbonate/ABS blends, ABS,polyvinyl chloride, polystyrene, polypropylene oxide, acrylics,polybutylene terephthalate (PBT), polyethylene terephthalate (PET),PBT/PET blends, nylons, blends of nylons, polyalkylene materials,fluoropolymers, cyclo-olefin polymers, or combinations thereof.According to various embodiments, the material of the substrate is acyclic olefin copolymer, for example, ZEONEX, available from ZEONCorporation, Tokyo, Japan, or TOPAZ, available from Ticona GmbH,Frankfurt, Germany.

The entire substrate can include an inelastically deformable material.According to various embodiments having a valve that includes anintermediate wall, at least the intermediate wall can include aninelastically deformable material. The intermediate wall need not beinelastic, but can be sufficiently non-elastic and deformable to enablethe formation of a fluid communication between two recesses that theintermediate wall separates upon deformation of the intermediate wall.According to various embodiments, the substrate can include a materialthat can withstand thermal cycling at temperatures of between 60° C. and95° C., as for example, are used in polymerase chain reactions.Furthermore, the substrate material can be sufficiently strong towithstand a force necessary to achieve manipulation of a fluid samplethrough the microfluidic device, for example, centripetal forcenecessary to spin and manipulate a sample within and through the device.

The substrate can include one or more base layers in contact with arecess-containing layer. The recess-containing layer can be a layerhaving holes formed therethrough, and a base layer can contact therecess-containing layer and define bottom walls of the through-holes inthe recess-containing layer. The substrate can have the same dimensionsas the microfluidic device and can make-up a major portion of thethickness of the microfluidic device.

According to various embodiments, the microfluidic device can beprovided with an elastically deformable cover layer that at least coversportions of the recess-containing substrate in areas where a portion ofthe substrate is to be deformed. For example, the cover layer can coverany number of a plurality of chambers or channels formed in thesubstrate, or cover all of the chambers and channels formed in thesubstrate. The cover layer can partially cover one or more chambers,input openings, output openings, columns, or other features formed in oron the substrate. The cover layer can have elastic properties thatenable it to be temporarily deformed when a deformer contacts the deviceand deforms an intermediate wall, for example, an intermediate walllocated underneath the cover layer. Once such a deformer is removed fromcontact with the microfluidic device, the deformable intermediate wallcan remain in a deformed state while the cover layer elasticallyrebounds, for at least an amount of time sufficient to enable fluidtransfer between two or more recesses that are fluidly connected bydeformation of the intermediate wall. The deformable material of theintermediate wall can be elastic to some extent, or can be inelastic.

The elastically deformable cover layer, and/or the substrate, can bechemically resistant and inert. The elastically deformable cover layercan include a material that can withstand thermal cycling attemperatures of between about 60° C. and about 95° C., for example, areused in polymerase chain reactions. Any suitable elastically deformablefilm material can be used for the cover layer, for example, elastomericmaterials. According to various embodiments, PCR tape materials can beused as or with the elastically deformable cover layer. Polyolefinmaterial films, other polymeric films, copolymeric films, andcombinations thereof can be used for the cover layer.

The cover layer can be a semi-rigid plate that bends over its entirewidth or length or that bends or deforms locally. The cover layer can befrom about 10 micrometers (μm) to about 500 μm thick, for example, 50 μmto 100 μm, and can include, an adhesive layer. If used, the adhesivelayer can be from about 50 μm to about 100 μm thick. Other materials,features, and aspects of the microfluidic devices, device substrates,device cover layers, and device walls, are described in U.S. ProvisionalPatent Application No. 60/398,851 to Bryning et al., which isincorporated herein in its entirety by reference.

FIG. 26 depicts a microfluidic device processing system 399 thatincludes a platen 380 that revolves around an axis of rotation 386,holders 381 and 383 for holding and securing the respective microfluidicdevices such as the devices shown in FIGS. 20 and 21, a heating element388, control unit 390. The processing system also includes a drive unit(not shown), and a control unit (not shown) for the drive unit. FIG. 26shows a direction of rotation with the unmarked arrow, however, thedirection of the rotation can be in the opposite direction instead.

FIGS. 27a-27d are cross-sectional views of various channel profiles thatcan be used in microfluidic devices according to various embodiments. InFIG. 27a , channel 542 is formed with a rectangular cross-sectional areain a substrate 540. The cross-sectional area can have an aspect ratio,that is a width/depth ratio of greater than one. In FIG. 27b , channel546 is formed with a semi-oval cross-sectional area in a substrate 544.The cross-sectional area can have an aspect ratio, that is, awidth/depth ratio of greater than one. In FIG. 27c , a thin and narrowchannel 550 is formed in a substrate 548, wherein the cross-sectionalarea can have an aspect ratio, that is, a width/depth ratio of less thanone. In FIG. 27d , a channel 554 is formed with a trapezoidalcross-sectional area in a substrate 552. These and other cross-sectionaldesigns can be used as flow-restricting channels and can be preformed orformed during a valve-opening operation according to variousembodiments.

The dimensional characteristics of a typical, straight channel flowrestrictor cross-section can be, for example, about 0.2 mm by about 0.2mm. The length of such a channel can be, for example, from about 0.1 mmto about 10 cm, for example, about 5 mm. A flow restrictor can be usedin conjunction with a larger chamber, greater than approximately 0.50mm, and serve to retain particles, for example, P-10, SEIE beads,particulates, and SEC beads, located in a chamber. The flow restrictorcan be located downstream of the chamber holding the particles.Downstream means the flow restrictor is located at a greater distancefrom an axis of rotation than the chamber. When subjected to acentripetal force, the materials in the chamber can move toward the flowrestrictor where the particulates can be retained while the fluids canpass into an adjacent channel or chamber.

According to various embodiments and as described above, dimensions ofthe flow restrictor are not limited to square cross-sections. Othershapes can be successfully implemented. For example, a rectangular flowrestrictor cross-section having a 0.10 mm depth and a 0.30 mm width canbe formed in a substrate to retain gel filtration media such as P-10beads available from BioRad.

According to various embodiments, the processing system can includemicrofluidic device holders on the platen that orient parallel pathwaysof the microfluidic devices off axis with regard to the axis of rotationof the platen. According to various embodiments, a holder can beprovided that aligns all of the parallel pathways of a microfluidicdevice such that when the pathways are parallel to a radius of theplaten all of the pathways lie off of the radius and on the same side ofthe radius.

According to various embodiments, a sample processing system is providedthat includes a microfluidic device, having a plurality of parallelpathways disposed in the holder, wherein each input opening of theplurality of pathways is closer to the axis of rotation than eachrespective output opening of the plurality of pathways. According tovarious embodiments, each of the plurality of parallel pathways of thedevice includes a respective input opening, at least one processingchamber, and output opening in a linear arrangement.

According to various embodiments, the microfluidic device used with thesample processing system is shaped as a rectanguloid having a length, awidth, and a thickness, and the holder is capable of holding themicrofluidic device securely to the platen. Clips, fasteners, or otherholding mechanisms can be employed to secure the device to the platen.According to various embodiments, a sample processing system is providedwhere the microfluidic device has opposing first and second rectangularsurfaces, where each of the surfaces has a length that is greater thanthe width thereof. According to various embodiments, a sample processingsystem is provided wherein a microfluidic device is disposed in theholder, and a radius of the platen is normal to the length of themicrofluidic device and wherein the device includes parallel pathwaysthat extend parallel to the length or the width of the device. Accordingto various embodiments, a sample processing system is provided wherein amicrofluidic device is disposed in the holder, and a radius of theplaten is normal to the width of the microfluidic device and wherein thedevice includes parallel pathways that extend parallel to the length orthe width of the device.

A description of other materials components and methods useful forvarious features of the microfluidic devices, systems, and methodsdescribed herein, is provided in U.S. Provisional Patent Application No.60/398,851 to Bryning et al., which is incorporated herein in itsentirety by reference.

The foregoing described and other sample processing devices can beprocessed alone. According to various embodiments, a sample processingdevice 610 can be mounted on a carrier 680. Such an assembly is depictedin an exploded perspective view of sample processing device 610 andcarrier 680 shown in FIG. 28.

By providing a carrier that is separate from the sample processingdevice, the thermal mass of the sample processing device can beminimally affected as compared to manufacturing the entire sampleprocessing device with a thickness suitable for handling with automatedequipment, for example, by robotic arms, and/or processing withconventional equipment. Another potential advantage of a carrier is thatthe sample processing devices may exhibit a tendency to curl orotherwise deviate from a planar configuration. Attaching the sampleprocessing device to a carrier can retain the sample processing devicein a planar configuration for processing. According to variousembodiments, the carrier can be made of plastic or other rigid materialto provide the carrier with sufficient rigidity when attached to thesample processing device. The plastic carrier can be provided with arubber pad or rubber pads attached to at least one surface thereof. Asilicone foam pad or layer can be used on a surface of the carrier, forexample, on the surface that contacts the sample processing device.

The carrier can be provided with limited areas of contact with thesample processing device to which it is mounted, to reduce thermaltransmission between the sample processing device and the carrier. Thesurface of the carrier facing away from the sample processing device canprovide limited areas of contact with, for example, a platen or otherstructure used to force the sample processing device against a thermalblock to reduce thermal transmission between the carrier and the platenor other structure. The carrier can have a relatively low thermal massto avoid influencing temperature changes in the sample processingdevice.

According to various embodiments, the carrier can exhibit somecompliance such that the carrier and/or attached sample processingdevice can conform to the surfaces between which the assembly iscompressed, for example, a thermal block and platen. Carriers themselvesmay not be perfectly planar due to, e.g., variations in manufacturingtolerances, etc. Further, the assemblies may have different thicknessesdue to thickness variations in the carrier and/or the sample processingdevice.

According to various embodiments, the sample processing device 610 canbe loaded using centripetal forces. The carrier can maintain theintegrity of the sample processing device by applying pressure to thecard during loading and/or thermal cycling.

The carrier 680 can be attached to the sample processing device 610 in amanner that allows for the carrier 680 to be reused with many differentsample processing devices 610. According to various embodiments, thecarrier 680 can be permanently attached to a single sample processingdevice 610 such that, after use, both the sample processing device 610and the carrier 680 are discarded together.

In the depicted embodiment, the sample processing device 610 includesmolded posts 611 for aligning the sample processing device 610 to thecarrier. At least one of the molded posts can be located proximate acenter of the sample processing device 610. Although only one moldedpost 611 can be used for attaching the sample processing device 610 tothe carrier 680, at least two posts 611 can be included. Thecentrally-located post 611 can assist in centering the sample processingdevice 610 on the carrier 680, while a second post 611 can be providedto prevent rotation of the sample processing device 610 relative to thecarrier 680. Further, although only two posts 611 are depicted, it willbe understood that three or more posts or other sites of attachmentbetween the sample processing device 610 and the carrier 680 can beprovided. Further, the posts 611 can be melt bonded to the sampleprocessing device 610 to accomplish attachment of the two components inaddition to alignment.

Posts or other alignment features can be provided on either or both ofthe sample processing device 610 and the carrier 680 to generally alignthe sample processing device 610 with the carrier 680 before the finalalignment and attachment using molded posts 611 on the sample processingdevice 610. The posts and/or other alignment features can align theassembly including the sample processing device 610 and carrier 680relative to, for example, a thermal processing system used to thermallycycle materials in the sample process chambers 650. One or morealignment features can also be used in connection with a detectionsystem for detecting the presence or absence of a selected analyte inthe process chambers 650.

According to various embodiments, posts or other alignment mechanismscan be provided on the carrier 680 to align the carrier 680 with athermal block. The posts can be arranged as cone-shaped or tapered pinsthat can mate with corresponding truncated or non-truncated cone-shapedor tapered wells or recesses formed in the thermal block. The posts canbe arranged to have a cross-like cross-section, such as a philips headscrewdriver tip, that can be compressible and/or elastic, and that canmate with cone-shaped or tapered wells or recesses formed in the thermalblock. The posts of the carrier 680 can be made of polypropylene. Thewells or recesses formed in the thermal block can have the shape of atruncated cone.

The carrier 680 can include various features such as openings 682 thatare preferably aligned with the process chambers 650 of the sampleprocessing device 610. By providing openings 682, the process chambers650 can be viewed through the carrier 680. One alternative to providingthe openings 682 is to manufacture the carrier 680 of a material (ormaterials) transmissive to electromagnetic radiation in the desiredwavelengths. The carrier 680 can be continuous over the surface of thesample processing device 610, that is, the carrier can be provided withno openings formed therethrough for access to the process chambers 650.

The sample processing device 610 and carrier 680 are exemplified in FIG.29, where it can be seen that the loading chambers 630 can extend beyondthe periphery of the carrier 680. As such, the portion of the sampleprocessing device 610 containing the loading structures 630 can beremoved from the remainder of the sample processing device 610 afterdistributing the sample material to the process chambers 650.

The carrier 680 illustrated in FIGS. 28 and 29 can also provideadvantages in the sealing or isolation of the process chambers 650during and/or after loading of sample materials in the process chambers650.

FIG. 30 is an enlarged view of a portion of the bottom surface of thecarrier 680, that is, the surface of the carrier 680 that faces thesample processing device 610. The bottom surface of the carrier 680includes a number of features including main conduit support rails 683that can extend along the length of the main conduits 640 in theassociated sample processing device 610. The support rails 683 can, forexample, provide a surface against which the main conduits 640 of thesample processing device 610 can be pressed while the conduit 640 isdeformed to isolate the process chambers 650 and/or seal the conduits640 as discussed above.

In addition to their use during deformation of the main conduits 640,the support rails 683 can also be relied on during, e.g., thermalprocessing to apply pressure to the conduits 640. Furthermore, the useof support rails 683 can also provide an additional advantage in thatthey provide for significantly reduced contact between the sampleprocessing device 610 and the carrier 680 while still providing thenecessary support for sealing of the main conduits 640 on device 610.

Contact between the carrier 680 and device 610 can be reduced orminimized when the assembly is to be used in thermal processing ofsample materials, for example as with polymerase chain reactions (PCR).As such, the carrier 680 can be characterized as including a carrierbody that is spaced from the sample processing device 610 between themain conduits 640 when the support rails 683 are aligned with the mainconduits 640. The voids formed between the carrier body and the sampleprocessing device 610 can be occupied by air or by, for example, acompressible and/or thermally insulating material. According to variousembodiments, the carrier 680 can be made from plastic and can have alayer of compressible foam attached to or abutting the surface facingthe sample processing device 610, for reducing thermal transmissionbetween the sample processing device 610 and the carrier 680. Accordingto various embodiments, the foam layer can be a silicone foam.

Also depicted in FIG. 28 are a number of optional compression structures684 which, in the exemplified embodiment, are in the form of collarsarranged to align with the process chambers 650 on the sample processingdevice 610. The collars define one end of each of the openings 682 thatextend through the carrier 680 to allow access to the process chambers650 on sample processing device 610. The compression structures 684, forexample, collars, are designed to compress a discrete area of the deviceproximate each of the process chambers 650 on the sample processingdevice 610 when the two components (the sample processing device 610 andthe carrier 680) are compressed against each other.

That discrete areas of compression can provide advantages such as, forexample, improving contact between the device 610 and the thermal blockproximate each of the process chambers. That improved contact canenhance the transfer of thermal energy into and/or out of the processchambers. Further, the improvements in thermal transmission can bebalanced by only limited thermal transmission into the structure of thecarrier 680 itself due, at least in part, to the limited contact areabetween the sample processing device 610 and the carrier 680.

Another advantage of selectively compressing discrete areas of thedevice 610 is that weakening of any adhesive bond, delamination of theadhesive, and/or liquid leakage from the process chambers 650 can bereduced or prevented by the discrete areas of compression. Thisadvantage can be particularly advantageous when using compressionstructures in the form of collars or other shapes that surround at leasta portion of the process chambers on the sample processing device.

The collars in the exemplified embodiment are designed to extend onlypartially about the perimeter of the process chambers 650 and are notdesigned to occlude the feeder conduit entering the process chamber 650.Alternatively, however, collars could be provided that are designed toocclude the feeder conduits, thereby potentially further enhancingisolation between the process chambers during thermal processing ofsample materials.

The collars 684 can optionally provide some reduction in cross-talkbetween process chambers 650 by providing a barrier to the transmissionof electromagnetic energy, for example, infrared to ultraviolet light,between the process chambers 650 during processing and/or analysis ofthe process chambers 650. For example, the collars 684 can be opaque toelectromagnetic radiation of selected wavelengths. Alternatively, thecollars 684 can inhibit the transmission of electromagnetic radiation ofselected wavelengths by diffusion and/or absorption. For example, thecollars 684 can include textured surfaces to enhance scattering, and/orthe collars 684 can include materials incorporated into the body of thecollar 684 and/or provided in a coating thereon that enhance absorptionand/or diffusion.

The carrier 680 can include force transmission structures to enhance thetransmission of force from the upper surface of the carrier 680, thatis, the surface facing away from the sample processing device, to thecompression structures, for example, in the form of collars 684 in theexemplary embodiment, and, ultimately, to the sample processing deviceitself.

FIG. 31 depicts a portion of an illustrative embodiment of a forcetransmission structure. The force transmission structure is provided inthe form of an arch 685 that includes four openings 682 and is operablyattached to collars 684. The force transmission structure defines alanding area 687 located between the openings 682 and connected to thecollars 684 such that a force 686 applied to the landing area 687 in thedirection of the sample processing device is transmitted to each of thecollars 684, and, thence, to the sample processing device (not shown).In the depicted embodiment, the landing areas are provided by the crownsof the arches 685.

The arch 685 can transmit the force evenly between the different collars684 attached to the arch 685, which are essentially provided as hollowcolumns supporting the arch 685 (by virtue of openings 682). This basicstructure is repeated over the entire surface of the carrier 680 as seenin, for example, FIG. 28.

Advantages of providing landing areas on the force transmissionstructures include the corresponding reduction in contact between thecarrier 680 and a platen or other structure used to compress the sampleprocessing device using the carrier 680. That reduced contact canprovide for reduced thermal transmission between the carrier 680 and theplaten or other structure used to compress the sample processing device.In addition, the force transmission structures and correspondingcompression structures on the opposite side of the carrier can allcontribute to reducing the amount of material in the carrier 680,thereby reducing the thermal mass of the carrier 680 and, in turn, theassembly of the carrier 680 and a sample processing device.

FIG. 32 illustrates another optional feature of carriers used inconnection with the present invention. The carrier 680′ is depicted withan optical element 688′, for example, a lens, that can assist infocusing electromagnetic energy directed into the process chamber 650′or emanating from the process chamber 650′. The optical element 688′ isdepicted as integral with the carrier 680′, although it should beunderstood that the optical element 688′ can be provided as a separatearticle that is attached to the carrier 680′.

FIG. 33 illustrates yet another optional feature of carriers that can beused. The carrier 680″ includes an alignment structure 687″ that can beused to assist guiding a pipette 611″ or other sample material deliverydevice into the appropriate loading structure on the sample processingdevice 610″. The alignment structure 687″ can be removed with theloading structures on the sample processing device 610″ as describedherein. The alignment structure 687″ can be generally conical asdepicted to guide the pipette 611″, if it is slightly off-center from aninlet port, into the loading structure on sample processing device 610″.

As an alternative the molded carrier depicted in FIGS. 28-31, thecarrier can be in the form of a sheet of material in contact with oneside of the sample processing device. FIG. 34 is an exploded view of oneillustrative sample processing device 710 and a carrier 780 that can beused in connection with the sample processing device 710.

The sample processing device 710 includes a set of process arrays 720,each of which includes process chambers 750 that, in the depicted sampleprocessing device 710, are arranged in an array on the surface of thesample processing device 710. The carrier 780 includes a plurality ofopenings 782 formed therein that preferably align with the processchambers 750 when the sample processing device 710 and carrier 780 arecompressed together.

The carrier 780 can be manufactured of a variety of materials, althoughit can be preferred that the carrier be manufactured of a compressiblematerial, for example, a sheet of compressible foam or other substance.In addition to compressibility, the compressible material can exhibitlow thermal conductivity, low thermal mass, and/or low compression set,particularly at temperatures to which the sample processing device maybe subjected. One class of suitable foams can include, for example,silicone based silicone foams.

If the carrier 780 is manufactured from compressible material, there maybe no need to provide relief on the surface of the carrier 780 facingthe sample processing device 710 to prevent premature occlusion of theconduits in the process arrays 720. If, however, the carrier 780 ismanufactured of more rigid materials, it can be desirable to providesome relief in the surface of the carrier 780 for the conduits in theprocess arrays 720.

Similar to the carrier 680 described above, a carrier 780 such as thatdepicted in FIG. 34 can provide selective compression of the sampleprocessing device by not compressing the sample processing device inareas occupied by process chambers 750 due to the absence of materiallocated above the process chambers 750. As a result, the carrier 780 canprovide several additional advantages. For example, the weakening of theadhesive bond, delamination of the adhesive, and/or liquid leakage fromthe process chambers 750 can be reduced or prevented by the compressionapplied to the sample processing device 710 surrounding the processchambers 750. In addition, thermal leakage from, for example, a thermalblock against which the assembly can be urged, can be reduced if thematerial of the carrier 780 is provided with desirable thermalproperties, for example, low thermal mass, low thermal conductivity, andthe like.

According to various embodiments, openings 782 can provide protectionfrom cross-talk between process chambers 750 by providing a barrier tothe transmission of electromagnetic energy, for example, light, betweenthe process chambers 750 during processing and/or analysis of theprocess chambers 750. For example, the carrier 780 can be opaque and/ornon-transmissive of electromagnetic radiation of selected wavelengths.Alternatively, the carrier can inhibit the transmission ofelectromagnetic radiation of selected wavelengths by diffusion and/orabsorption. For example, the openings 782 can include textured surfacesto enhance scattering. Moreover, the carrier 780 can include materialsincorporated into the body of the carrier 780, and/or provided in acoating thereon, that can enhance absorption and/or diffusion ofselected wavelengths of electromagnetic energy.

According to various embodiments, the carriers described above inconnection with FIGS. 28-34 can be fixedly attached to the sampleprocessing device, or the carriers can be separate from the sampleprocessing device. If separate, the carriers can be removably attachedto, or brought into contact with, each sample processing device in amanner that facilitates removal from an sample processing device withoutsignificant destruction of the carrier. As a result, the carrier can beused with more than one sample processing device. Alternatively, thecarrier can be firmly affixed to the sample processing device, such thatboth components can be discarded after use. In some instances, thecarrier can be attached to the system used to process the sampleprocessing device, for example, a platen of a thermocycling system, suchthat as a sample processing device is loaded for thermal processing, thecarrier can be placed into contact with the sample processing device.

Both of the carriers described above are examples of means forselectively compressing together the first side and the second side of asample processing device, about each process chamber. The compressioncan occur simultaneously about each process chamber. Many otherequivalent structures that accomplish the function of selectivelycompressing the first side and second side of a sample processing devicetogether about each process chamber can be envisioned by those of skillin the art. In some configurations, the means for selectivelycompressing, for example, the resilient carrier 780, can applycompressive force over substantially all of the sample processing deviceoutside of the process chambers. In other embodiments, the means forselectively compressing can apply compressive forces in only a localizedarea about each of the process chambers in the sample processing device,for example, carrier 680 with its associated collars.

Any system incorporating a means for selectively compressing can be usedto attach the means for selectively compressing to the sample processingdevice or to a platen or other structure that is brought into contactwith the sample processing device during processing. FIG. 35 depicts onethermal processing system that can be used in connection with the sampleprocessing devices in a block diagram format. The system includes asample processing device 710′ located on a thermal block 708′. Thetemperature of the thermal block 708′ is preferably controlled by athermal controller 706′. On the opposite side of the sample processingdevice 710′, the means for selectively compressing, in the form ofcarrier 780′, is located between the sample processing device 710′ and aplaten 704′. The platen 704′ can be thermally controlled, if desired, bya thermal controller 702′ that can, in some instances, be the same ascontroller 706′ controlling the temperature of the thermal block 708′.The sample processing device 710′ and the means for selectivelycompressing 780′ can be compressed between the platen 704′ and thermalblock 708′ as indicated by arrows 701′ and 702′ during thermalprocessing of the sample processing device 710′.

Those skilled in the art can appreciate from the foregoing descriptionthat the broad teachings herein can be implemented in a variety offorms. Therefore, while the devices, systems, and methods herein havebeen described in connection with particular embodiments and examplesthereof, the true scope of the present invention should not be solimited. Various changes and modifications may be made without departingfrom the scope of the present invention, as defined by the appendedclaims.

What is claimed:
 1. A microfluidic device comprising: a substrate havinga first surface, a second surface opposing the first surface, and athickness; a plurality of parallel pathways formed in the substrate,each of the pathways comprising an input opening, an output opening, atleast one processing chamber located between the input opening and theoutput opening, wherein the input opening, the at least one processingchamber, and the output opening are arranged linearly; a first fluidcommunication between the input opening and the at least one processingchamber, and a second fluid communication between the at least oneprocessing chamber and the output opening; wherein each of the pluralityof pathways includes at least one valve that is capable of being openedto form a fluid communication, and the at least one valve comprises afirst deformable material having a first elasticity, and a seconddeformable material having a second elasticity that differs from thefirst elasticity.
 2. The microfluidic device of claim 1, wherein atleast one of the first and second fluid communications includes achannel formed in the first surface; and the other of the first andsecond fluid communications includes a channel formed in the secondsurface.
 3. The microfluidic device of claim 1, further comprising afirst cover in contact with the first surface of the substrate, andwherein the first cover is the second deformable material.
 4. Themicrofluidic device of claim 1, further comprising a size exclusionfiltration material disposed in the at least one processing chamber. 5.The microfluidic device of claim 1, further comprising components forenabling polymerase chain reaction of a nucleic acid sequence, disposedin the at least one processing chamber.
 6. The microfluidic device ofclaim 1, wherein the at least one processing chamber is shaped as achannel in the first surface of the substrate.
 7. The microfluidicdevice of claim 1, wherein the substrate is rectangular.